Capillary Electrophoresis (CE) Troubleshooting Tips:

What follows is a short list of problems, "observations" followed by a list of areas that should be investigated, as appropriate in parenthesis (), to troubleshoot common problems seen when using the analytical technique of capillary electrophoresis, CE, CZE.

 Observation (Investigate for cause):

            Excessive Baseline Drifting up or down

·         Temperature is not stable (stabilize room and/or capillary temperature).

·         Fouling of capillary (replace or clean and wash capillary with fresh, filtered solution).

·         Current levels unstable (loose connections, partial obstruction in capillary or running out of buffer solutions).

·         Capillary may have poorly cut ends resulting in poor connections or flow (replace capillary).

Excessive Signal Noise

·         Detector has air in flow cell (purge capillary and wash flow path).

·         Current level may be too high (reduce current).

·         Detection parameters, wavelength and bandwidth, may be inappropriate for buffer solution (select appropriate detection settings which are appropriate for the buffer used and selective for the analyte).

Loss of Signal

·         Voltage/Current has turned off (turn ON or investigate if system is in “alarm” state due to an error).

·         Detector parameters not selected.

·         Capillary has not been fully equilibrated (equilibrate capillary and auto-zero the scale).

·         Baseline offset may be off-scale (after equilibration, adjust scale or auto-zero).

·         Detector lamp(s) off, not ignited or due for replacement (verify lamp operation).

Signal Peak Shape Issues

·         Truncated, clipped or ‘square’ peaks (sample overload, reduce concentration 10x, shorten load time and re-evaluate).

·         Tailing peaks often result from very high current or when the concentration of buffer is too high (lower the current and/or reduce the buffer concentration, then re-evaluate).

·         Sampling rate may be too low (measure the peak width in units of time (i.e. seconds), then configure the detector to insure that the sampling rate allows for at least 20 points to be collected per average peak width (30 points is a better target # to use).

·         No peaks observed (Many possible causes, including: Partially or fully obstructed capillary, broken capillary, out of buffer, no injection, detector settings inappropriate for analysis, current too low, pressure too low. Look for a small peak from the injection along the start of the baseline to confirm that an analysis was started, then troubleshoot the method and settings).

            General Stability and Noise Issues 

·   When the CE system has not been used in a few days, salts from the buffer solution(s) may deposit on and clog the capillary line, flow cell and/or sensors. To avoid these problems, be sure to thoroughly clean, flush and wash down the flow path before use. Take the time to prepare fresh filtered solutions (each day) and allow time for the system to equilibrate. Taking these basic steps will avoid many hours/days of frustration.

Cannabis, Cannabinoid (Hemp, THC, CBD, CBN, Marijuana) HPLC Analysis and Testing, Areas for Improvement [*Updated 8/2021]

Over the past few years we have observed an exponential growth in the number of state-level, legal businesses (in the USA) who offer Cannabis Analysis (e.g. Potency Analysis or Profiles) and/or related businesses such as Hemp Oil Extraction. Most related products which incorporate Marijuana, CBD, THC, Cannabinoids, Terpenes and/or other related compounds require formal analytical laboratory testing which should follow good laboratory practices. This article is targeted to help many of the people involved in this new analysis business (or anyone using chromatography as the analytical technique of choice for the same goals).

As a professional chromatography consultant, I have seen a large increase in the number of requests for my services to this new market. Most of these new businesses have questions about obtaining professional training, correct analysis procedures, improving reproducibility, documentation, optimizing method development, how to receive professional training in maintenance of the HPLC system(s) and need hands-on help to optimize the procedures used. Many users are not achieving acceptable results and need help finding out why. They want to know where they can take a class to learn HPLC method development and how to perform the required tests. 

These new businesses would benefit greatly from professional guidance EARLY in their setup and establishment, to improve the internal methods and procedures of analysis used. Time spent on the "front-end" of any process is always time spent wisely (in this context, knowledge and practical experience = confidence). A chromatography professional can quickly identify areas which may need improvement and/or suggest changes that can directly improve your company's accuracy, reproducibility, increase efficiency and of course, impact your bottom-line too. Focus is placed on the exact areas that will benefit you (rather than wasting time with non-targeted approaches, sales biased classes and trial-and-error approaches). 

• Please note that there are NO SCIENTIFICALLY LEGITIMATE ONE DAY, ONE WEEK or ONE MONTH LONG TRAINING CLASSES THAT CAN TEACH YOU HOW TO PERFORM HPLC METHOD DEVELOPMENT or ANALYSIS. NONE AT ALL. Most types of "Certification" offered are completely without scientific merit or value. The training needed takes many years of hands-on experience, in an industrial laboratory (not a school), to acquire just a basic level of proficiency (*Emphasis on 'Basic", not intermediate or expert). Be very cautious of anyone who claims to be able to provide you with all the training you need in a short time period.
  

Generating accurate and reproducible analytical data, esp. with HPLC, SFC or GC requires a great deal of knowledge, formal training and practical hands-on experience (not something which is taught at most university or school programs). These complex techniques require years of bench time and professional hands-on experience to learn). Shipping or selling products which contain unacceptable levels of impurities or which do not meet basic testing or regulatory qualifications could pose a health and/or liability risk. Hire people who have the needed training from industry before setting up the laboratory.

It has been my professional experience that some of the most common training areas that client's would benefit from are: GLP (Good Laboratory Practices/Procedures and SOPs) and additional instrument operational training to demonstrate proficiency in analytical chromatography. Address these areas early on and continuously update them to reduce errors and improve results. Training should continue on a regular basis to gain experience.
 
While each confidential consultation visit I have with a client may show different key issues which need to be addressed, many labs can start to improve their analytical results by addressing and improving how they address:

1. Documentation: Laboratory methods and sample analysis must be conducted using clearly written documentation. This should initially include having Standard Operating Procedures (SOPs) in place for all methods, procedures, qualification, verification and tests used. They should include SOPs, Document Control and Policy documents which also address: Training, Calibration, Maintenance, Frequency of the same, Mobile phase preparation, pH measurement, use of the balance and so on... I find that it is best to create an initial SOP Template to insure document uniformity (i.e. include such fields as: Business Name, SOP #, review/approval date(s)/names, Rev #, materials & tools needed, purpose, procedure steps, pass/fail definitions... plus any needed supporting documents).
2. Sample Preparation Methods: Be sure to document, test, review / standardize specific sample preparation methods, for each sample type. Variations in: temperature, extraction solvent or the solution(s) dissolved in, homogenization or grinding methods, mixing, times used, glass or plastic containers used may result in significant variation of the final reported results.
3. Correct Poor Reproducibility and/or Baseline Instability Issues: In chromatography analysis, if the method(s) used are not stable and reproducible, every time they are run, then little to no scientific value can be obtained from them. Methods used must follow basic good chromatography fundamentals and meet basic guidelines. Baseline noise or instability may directly impact integration results (which directly impacts reported results). Instrumentation must be operated in clean, climate controlled rooms. Failure to reproduce a result within acceptable limits (these will vary per method type) will invalidate the method used. Make sure that SOP's are followed, mobile phase solutions are made fresh each day (do not pre-mix solutions with acids and let them sit for several days before use; do not "top off" bottles), solutions should be degassed, HPLC columns are properly washed and re-equilibrated before each analysis, instruments are maintained (per a SOP) and serviced on a regular basis. 
4. Develop HPLC methods that follow good chromatography fundamentals: Retain, separate and resolve ALL peaks. Insure peak K primes are 2.0 or higher. If you have co-eluted peaks in your method, then method development is not finished. If you have ghost peaks or changing retention times, then you need to stop running samples and find out why. Be careful whose method(s) you use. A method that is "Validated" may not be scientifically valid method to use. Have the method checked by an experienced chromatographer.
   
5. Continuous Training is Required to become Proficient: To be proficient, at a basic level in chromatography, takes most chromatographers several years working in an industrial environment to gain practical hands-on time. That assumes that they have had professional training outside of college, in an industrial lab, and can demonstrate an understanding of the fundamentals of good chromatography. Note, that method development skills require a much higher level of understanding and hands-on training to acquire the needed skill set. Make sure your scientists have the needed level of training to operate, run analysis methods and troubleshoot any issues that come up (and issues will come-up, even under ideal conditions). Please do not make the mistake of thinking they will "figure it out" on their own. Hire people who already have several years of industrial chromatography experience, then provide them with additional training opportunities to advance their skills in the application areas that your business needs.  Get them help NOW, you will save money and time, plus get back on track moving forward with your project.
   

If you want to surpass your competitors and provide clients with the most accurate data, then investing in your employees professional knowledge and hands-on technical training is the fastest route to do so. This is an experience based technique where decades of practical knowledge are needed to improve your skill set. A professional can quickly provide you with practical information and show you techniques that will help you move forward. 1-2 days of on-site training often translates to nearly one years worth of knowledge. What is one-years worth of lost time worth to you?
  
Additional Resources:

• Modern HPLC Method Development Tips (PART I): 
• Modern HPLC Method Development Tips (PART II):
• EXPERT CANNABINOID HPLC Training and CONSULTING SERVICES: 

The Three Most Common HPLC Questions and How To Solve Them

The three most common HPLC related questions I am asked each week can be summarized below. Test your basic chromatography knowledge. Before reading the answers, see if you can answer them correctly on your own.

• "What Is Causing the HPLC Baseline, Pressure or Peak Retention Time(s) To: Wander, Change, Drift, Vary or be Unstable?"

• "How Should I Wash or Regenerate My HPLC Column?"

• "How Can I Tell if the Sample is Retained On the HPLC Column? or What Does It Mean When No Chromatography Took Place?"

Let us address each question in order and attempt to provide accurate answers (I have included links after each question to articles with more detailed explanations).

What Is Causing the HPLC Baseline, Pressure or Peak Retention Time(s) To: Wander, Change, Drift, Vary or be Unstable?

• Retention times must be reproducible from run to run.The causes of an unstable baseline and/or changing peak retention time(s) are often related. Common reasons include: Column temperature fluctuations, inadequate mobile phase mixing or degassing, leaks, dirty column, sample overload, lack of pH or buffering control (weakly ionizable samples can be very sensitive to changes). *Full Article link with detailed answers, here.

How Should I Wash or Regenerate My HPLC Column?

Note: Before proceeding with any column regeneration or cleaning procedures, always refer to the specific advice provided by the column manufacturer. Approved maintenance and cleaning instructions can often be found in the product guide or booklet which comes with the new column. Additional information can be found on the vendor's website or by contacting them directly.

• Two issues must be addressed to answer these types of questions. (1) Always wash your column with a specific column wash solution which is stronger than your analysis solution. The use of a stronger solution (In this context, "stronger" means better at dissolving the samples and faster at eluting them from the column) as the wash solution requires regular use to maintain the column. Failure to regularly wash your column may result in compounds accumulating on the column over time (fouling the column) resulting in poor reproducibility, higher back-pressures, contamination and/or poor peak shape. (2) Next, always wash your column after each analysis. This should be a separate step, not incorporated into your analysis method. The analysis method should not include the column re-equilibration steps at all. A second, separate wash method should always follow each analysis method which includes the rinsing of the column with a "stronger" solution for an adequate period of time, then adjustment back to initial conditions where re-equilibration can take place to get it ready for the next analysis run. These are fundamental guidelines of good method development and follow well established principles. Developing methods in this way should increase the lifetime of your columns and improve the reproducibility of results obtained (better %RSD run-to-run).

For more information on washing bound proteins off RP HPLC columns, please refer to this linked article found here.

How Can I Tell if the Sample Is Retained On the HPLC Column? or What Does It Mean When the Sample Comes Out At or Near the Column Void Volume?

• Chromatography is a tool which when used properly adds one or more additional dimensions of physical or chemical characterization information to your analysis data. It does so first by using on-column RETENTION. Samples must be run under conditions which allow the material to interact with the chromatography support for a period of time. We define this time as the retention time. A sample which does not interact at all with the column support material will elute off the column early (and not be retained) at the "column void time" (or column dead time). We refer to this void time as the "T zero" time. When a sample elutes at or near the T zero time, no chromatography has taken place and no method has been developed. It is as if the HPLC column was not used. How do you know what the "T zero" time is (it will be different for different methods)? You must first calculate the HPLC column's dead volume. Once you know the column dead volume and flow rate, you can calculate the T zero time. A scientifically valid HPLC method will include conditions which retain the sample on the column for a long enough period of time to insure that it is interacting with the support. This allows for separation from other compounds to take place and is the purpose of chromatographic resolution. Without this retention mechanism, you are just flow-injecting the sample past the column and skipping all chromatography. It would be far simpler to just place the sample in a spectrophotomer cell as no retention or additional data would be obtained using that technique.

• When first learning liquid chromatography, two of the very first calculations you must learn to use in HPLC are: Column Dead Volume (aka: Column Void Volume) and the K prime of a sample (aka: Peak Capacity Factor). Do you know how to calculate these? They are calculated and reported for each method used. You should be able to tell anyone who asks you what the values are for each method. A chromatographer must know and understand them before using an HPLC system or running a method. They are also critical to method specificity and proper validation. Here are links which after reading and practicing, should make you an expert in these two fundamental calculations. 

"Determination of HPLC Column Dead Volume / Dead Time (T zero)";

"K Prime (also known as: Capacity Factor or Retention Factor): One of the Single Most Important HPLC Parameters of All".

So, how did you do answering these basic questions? If you have put in the needed study time and practical experience to learn and use these fundamentals of high-performance liquid chromatography, then you should have been able to easily provide correct answers to all three questions. If not, then it is time to go back and study up on those basic liquid chromatography texts and article links, plus get more supervised hands-on time with the instruments.